BK channel properties correlate with neurobehavioral severity in three KCNMA1-linked channelopathy mouse models

KCNMA1 forms the pore of BK K+ channels, which regulate neuronal and muscle excitability. Recently, genetic screening identified heterozygous KCNMA1 variants in a subset of patients with debilitating paroxysmal non-kinesigenic dyskinesia, presenting with or without epilepsy (PNKD3). However, the relevance of KCNMA1 mutations and the basis for clinical heterogeneity in PNKD3 has not been established. Here, we evaluate the relative severity of three KCNMA1 patient variants in BK channels, neurons, and mice. In heterologous cells, BKN999S and BKD434G channels displayed gain-of-function (GOF) properties, whereas BKH444Q channels showed loss-of-function (LOF) properties. The relative degree of channel activity was BKN999S > BKD434G>WT > BKH444Q. BK currents and action potential firing were increased, and seizure thresholds decreased, in Kcnma1N999S/WT and Kcnma1D434G/WT transgenic mice but not Kcnma1H444Q/WT mice. In a novel behavioral test for paroxysmal dyskinesia, the more severely affected Kcnma1N999S/WT mice became immobile after stress. This was abrogated by acute dextroamphetamine treatment, consistent with PNKD3-affected individuals. Homozygous Kcnma1D434G/D434G mice showed similar immobility, but in contrast, homozygous Kcnma1H444Q/H444Q mice displayed hyperkinetic behavior. These data establish the relative pathogenic potential of patient alleles as N999S>D434G>H444Q and validate Kcnma1N999S/WT mice as a model for PNKD3 with increased seizure propensity.

Introduction KCNMA1-linked channelopathy encompasses an array of neurological symptoms associated with clinical detection of a KCNMA1 variant. Affected individuals typically present with epilepsy and/or dyskinesia, but also have other disorders including ataxia, developmental delay, intellectual disability, and brain and structural abnormalities (Bailey et al., 2019;Liang et al., 2019;Miller et al., 2021). The basis for these symptoms is not mechanistically established but is likely similar to other neurological channelopathies involving direct or indirect changes in neuronal excitability leading to excitationinhibition imbalance (Benatar, 2000;Menezes et al., 2020). KCNMA1 genotype-phenotype correlation is an active area of investigation with >40 variants identified in this patient population to date (Miller et al., 2021, and ALM unpublished data). Since most variants arise de novo in a single heterozygous proband, whether 'KCNMA1 channelopathy' is a bona fide monogenic disorder, or results from intergenic and developmental interactions, is not well understood. Animal models for the most common variants are needed to validate genotype-phenotype associations and to investigate disease mechanisms and manifestations over lifespan (MacArthur et al., 2014).
Two GOF KCNMA1 variants, D434G and N999S, account for half of the patient population (Bailey et al., 2019;Miller et al., 2021). Both variants cause BK channel activation at more negative membrane potentials, speed activation, and slow deactivation (Diez-Sampedro et al., 2006;Du et al., 2005;Li et al., 2018;Moldenhauer et al., 2020a;Wang et al., 2009;Yang et al., 2010). The majority of individuals harboring D434G and N999S variants present with paroxysmal non-kinesigenic dyskinesia (PNKD type 3; OMIM #609446), characterized by varying degrees of negative motor phenomena eLife digest So far, only 70 patients around the world have been diagnosed with a newly identified rare syndrome known as KCNMA1-linked channelopathy. The condition is characterised by seizures and abnormal movements which include frequent 'drop attacks', a sudden and debilitating loss of muscle control that causes patients to fall without warning.
The disease is associated with mutations in the gene for KCNMA1, a member of a class of proteins important for controlling nerve cell activity and brain function. However, due to the limited number of people affected by the condition, it is difficult to link a particular mutation to the observed symptoms; the basis for the drop attacks therefore remains unknown. Park et al. set out to 'model' KCNMA1linked channelopathy in the laboratory, in order to determine which mutations in the KCNMA1 gene caused these symptoms.
Three groups of mice were each genetically engineered to carry either one of the two most common mutations in the gene for KCNMA1, or a very rare mutation associated with the movement symptoms. Behavioural experiments and studies of nerve cell activity revealed that the mice carrying mutations that made the KCNMA1 protein more active developed seizures more easily and became immobilized, showing the mouse version of drop attacks. Giving these mice the drug dextroamphetamine, which works in some human patients, stopped the immobilizing attacks altogether.
These results show for the first time which specific genetic changes cause the main symptoms of KCNMA1-linked channelopathy. Park et al. hope that this knowledge will deepen our understanding of this disease and help develop better treatments. Each α subunit is comprised of seven transmembrane domains (S0-S6) and an intracellular gating ring with hydrophobic segments (S7-S10, black). Pore (+) opening and closing is regulated by voltage-sensitive residues in S2-S4 (VSD), the AC domain (βA to αC), and two Regulators of Conductance of Potassium (RCK) domains in the gating ring (gray), each containing a Ca 2+ binding site (green) (Yang et al., 2015;Giraldez and Rothberg, 2017). (B) BK channel Figure 1 continued on next page described as dystonia, hypotonia, non-narcoleptic cataplexy, and akinesia. PNKD3 episodes manifest as postural lapses with immobility ('drop attacks') that occur with short duration and high frequency, often hundreds of times per day (Du et al., 2005;Heim et al., 2020;Keros et al., 2022;Li et al., 2018;Wang et al., 2017;Zhang et al., 2015). Just under half of patients experience seizure of varying types, including absence, atonic, myoclonic, and generalized tonic-clonic (GTC). However, epilepsy and PNKD are not consistently co-morbid (Du et al., 2005;Miller et al., 2021). Individuals with putative LOF variants report additional movement disorders including dyskinesia, axial hypotonia, tremor, or ataxia, in addition to various seizure types (Du et al., 2020;Liang et al., 2019;Rodrigues Bento et al., 2021;Tabarki et al., 2016;Yeşil et al., 2018). It is not yet clear whether variations in symptomatic presentation result from incomplete or inconsistent clinical evaluations, or genuine genotype-phenotype differences within either GOF or LOF cohorts.
We address these questions through heterologous, neuronal, and neurobehavioral validation for three patient-associated KCNMA1 variants in mouse models. The GOF BK N999S and BK D434G channels produced increased neuronal BK currents and firing as heterozygous alleles in transgenic mice, while heterozygous LOF BK H444Q channels were insufficient to alter neuronal properties. Mice were evaluated in a series of spontaneous and evoked seizure and locomotor assays. N999S propagated the largest symptomatic burden with chemoconvulsant challenge and stress-triggered dyskinesia, supporting the conclusion that this variant has the greatest monogenic pathogenicity, followed by D434G, Kcnma1 -/-, and H444Q. The results identify Kcnma1 N999S/WT mice as a PNKD3 model with the highest phenotypic similarity to patients harboring KCNMA1 GOF variants, including symptomatic resolution with acute dextroamphetamine treatment. Our findings further establish the stress-induced PNKD assay to delineate distinct symptomatic manifestations between GOF and LOF alleles, supporting its utility in a battery of neurobehavioral evaluations to define KCNMA1-linked channelopathy models.
structure showing two opposing subunits with Ca 2+ bound in the gating ring (PDB 6V38). H444Q (purple) and D434G (blue) are located within the βB-αB and αA and βB of the AC domain, respectively, a region within RCK1 affecting Ca 2+ -dependent gating (Du et al., 2005;Tao and MacKinnon, 2019). N999S (red) is located at the helix bend in the middle of the S10 domain within RCK2 (Tao and MacKinnon, 2019). (C) Representative inside-out patchclamp recordings from BK WT , BK N999S , BK D434G , BK H444Q channels expressed in HEK293 cells. Macroscopic BK currents were recorded in symmetrical K + and 1 μM intracellular Ca 2+ by holding patches at −100 mV, stepping from −100 to 250 mV for 30 ms, followed by a tail step −100 mV for 15 ms. Scale bars: 1 nA, 5 ms. (D) Normalized conductance-voltage (G-V) relationships fit with Boltzmann functions (solid lines). There was no change in the slope factor (z) for any of the variants (p=0.06, one-way ANOVA). BK WT (n=12), BK N999S (n=9), BK D434G (n=12), and BK H444Q (n=12). (E) Voltage of half-maximal activation (V 1/2 ) obtained from Boltzmann fits for individual patches. *p<0.0001. One-way ANOVA with Dunnett's post hoc. (F) Activation time constants (τ act ). BK N999S and BK D434G channels had decreased τ act compared to BK WT , either across all voltage steps (mixed effects model for repeated measures with Bonferroni post hoc, p<0.01) or above 120 mV (p<0.05), respectively. At lower voltages, BK D434G channels were more steeply voltage dependent, but did not exceed the fast activation time constants of BK N999S channels. BK H444Q channels had increased τ act compared to BK WT between 160 and 250 mV (p<0.05). Inset: Representative current traces from 170 mV step, scaled to the maximal current to illustrate activation timecourse (x-axis scale bar: 10 ms). τ act for BK WT currents was 3.8±0.3 ms, while BK N999S and BK D434 currents activated faster (0.9±9.1 and 1.8±0.1 ms, respectively) and BK H444Q activated slower (6.5±0.5 ms). (G) Deactivation time constants (τ deact ). BK N999S and BK D434G channels had increased τ deact compared to BK WT , across all voltage steps (mixed effects model for repeated measures with Bonferroni post hoc, p<0.01), with the exception of -160 (p>0.05), respectively. BK H444Q channels had decreased τ deact compared to BK . Inset: Representative current traces from -20 mV step, scaled to the maximal current to illustrate deactivation timecourse (x-axis scale bar: 10 ms). τ deact for BK WT currents was 0.7±0.01 ms, while BK N999S and BK D434 currents deactivated slower (4.5±0.7 and 1.5±0.1 ms, respectively) and BK H444Q deactivated more quickly (0.4±0.01 ms). Data are presented as mean ± SEM. Additional data on the effects of stimulants on BK WT and BK N999S channels appears in

Results
Patient variants confer GOF (N999S and D434G) and LOF (H444Q) properties on BK channel activity A comparative assessment for three dyskinesia-associated patient variants (N999S, D434G, and H444Q) was performed within the human BK channel (Figure 1). BK channel function was assessed using inside-out patch-clamp recordings in HEK293 cells. Patches from cells expressing BK WT , BK N999S , BK D434G , BK H444Q channels were activated with depolarizing voltage steps, and the voltage dependence of activation and kinetics were assessed from macroscopic currents ( Figure 1C). Conductance versus voltage (G-V) relationships ( Figure 1D) were assessed by the voltage of half-maximal activation (V 1/2 ; Figure 1E).
BK WT currents had a V 1/2 of 125±2 mV. Introduction of N999S and D434G mutations shifted the G-V relationships to more negative membrane potentials (V 1/2 : BK N999S 41±3 mV and BK D434G 88±2 mV), confirming their GOF effect at all voltages. The decrease in V 1/2 for BK N999S channels compared to BK WT was 20-30 mV larger than observed in prior studies with different splice variants and intracellular Ca 2+ Moldenhauer et al., 2020a). Here, under equivalent conditions, N999S produced a larger hyperpolarizing shift from WT (ΔV 1/2 = 84 mV) versus D434G (ΔV 1/2 = 37 mV). In addition, N999S and D434G produced faster BK channel activation and slower deactivation compared to WT ( Figure 1F-G). Altogether, BK N999S channels showed greater GOF properties than BK D434G in all parameters under these conditions, corroborating the relative severity predicted from prior work.
In contrast, introduction of the H444Q variant shifted the G-V relationship to more positive potentials (V 1/2 : BK H444Q 155±4 mV), consistent with LOF effects. H444Q produced changes in channel opening and closing further consistent with LOF effects, slowing activation and speeding deactivation ( Figure 1D-G). H444Q produced a smaller difference from WT than either GOF variant (ΔV 1/2 = 30 mV), identifying H444Q as a comparatively mild variant. The results indicate that N999S produces the strongest effect on BK channel activation in the GOF direction, followed by D434G (GOF) and H444Q (LOF).

Generation of N999S, D434G, and H444Q mouse models
Correlation between patient genotype and phenotype has only been established for a single KCNMA1 variant so far, D434G, an autosomal dominant that co-segregates with PNKD and epilepsy in a multigeneration pedigree (Du et al., 2005). D434G pathogenicity is further corroborated by mouse and fly models, which show alterations in neuronal excitability, brain and motor function (Dong et al., 2021;Kratschmer et al., 2021). In contrast, N999S and H444Q lack this direct evidence due to the absence of familial transmission among the children that carry these variants (Miller et al., 2021). N999S is the most common de novo KCNMA1 variant (~17% of all patients), found as heterozygous in every case. About half of individuals harboring N999S alleles are diagnosed with seizure, PNKD, or both (Keros et al., 2022;Li et al., 2018;Wang et al., 2017;Zhang et al., 2015), suggesting a strong potential to be causative in channelopathy symptoms. H444Q is found in a single case and is one of several putative LOF variants where affected individuals have dyskinesia-like paroxysms (Miller et al., 2021). This proband had a history of abnormal EEG, unresolved with respect to the diagnosis of epilepsy, but also harbors three additional genetic findings (ALM unpublished data).
To establish genotype-phenotype correlations, heterozygous mice replicating the patient genotypes were first evaluated. Each variant was introduced as a single nucleotide mutation into the mouse Kcnma1 gene using CRISPR base-editing ( Figure 1-figure supplement 1). In all animal experiments, investigators were blinded to genotype during data collection and analysis, and WT controls were compared to transgenic littermates within individual transgenic lines. Kcnma1 N999S/WT , Kcnma1 D434G/WT , and Kcnma1 H444Q/WT mice were grossly behaviorally and morphologically normal with no notable spontaneous paroxysms, gait abnormalities, or visually detectable seizures during home cage observation. Kcnma1 D434G/WT and Kcnma1 H444Q/WT intercrosses produced homozygous progeny that were also visually normal. However, Kcnma1 N999S/WT intercrosses produced no homozygous pups (see Materials and methods). The absence of homozygous N999S progeny is similar to Tg-BK R207Q mice harboring another strong GOF mutation that showed lethality in the homozygous allele configuration (Montgomery and Meredith, 2012). Given the inability to generate homozygous N999S mice, gene expression was analyzed from hippocampus and cerebellum of Kcnma1 N999S/WT and WT littermates (n=3 mice each genotype and tissue). No significant differences were found in the levels of Kcnma1 (1.07-fold change, p=0.79, FDR = 0.99, ANOVA with eBayes test), or BKβ subunits expressed in brain: Kcnmb1 (−1.07, p=0.14), Kcnmb2 (1.00, p=0.84), and Kcnmb4 (−1.03, p=0.46). The results show no gross up-or down-regulation of BK channel components in either the hippocampus or cerebellum of Kcnma1 N999S/ WT neurons. However, since the microarray-based probe set does not distinguish between the WT and N999S Kcnma1 transcripts, no further conclusion can be made regarding the specific expression ratio of each allele.

N999S and D434G increase BK current in hippocampal neurons
From heterologous cells, we predicted that the variants would have a strong (N999S), intermediate (D434G), or weak (H444Q) potential to alter neuronal BK current levels in transgenic mice. However, heterozygous patient genotypes create the possibility for hetero-tetramer channel formation (Geng et al., 2021), necessitating understanding the relative GOF and LOF effects in vivo from BK current levels in heterozygous transgenics compared to WT littermates. Recordings were made in the dentate gyrus of the hippocampus, where BK channels are highly expressed, regulate neuronal excitability, and where changes in BK channel properties are associated with seizure (Kaufmann et al., 2010;Knaus et al., 1996;Misonou et al., 2006;Sailer et al., 2006;Sausbier et al., 2005;Sausbier et al., 2006, Trimmer, 2015. In dentate granule cells, excitability is sensitive to changes in BK current in both directions, assessed using pharmacological inhibition as well as deletion of the β4 subunit (Brenner et al., 2005;Mehranfard et al., 2014;Mehranfard et al., 2015). Loss of β4 creates GOF BK channels by speeding BK channel kinetics, and Kcnmb4 -/knockout mice have temporal lobe seizures (Jaffe and Brenner, 2018;Petrik et al., 2011;Wang et al., 2016;Whitmire et al., 2017).
In contrast, BK currents in Kcnma1 H444Q/WT neurons were not significantly different compared to WT littermates at any voltage (Kcnma1 WT/WT 10.9±1.0 pA/pF and Kcnma1 H444Q/WT 9.5±1.9 pA/pF at -10 mV; -13% change; Figure 2C). This establishes an allelic series of Kcnma1 D434G/D434G >> Kcnma1 D434G/WT ≈ Kcnma1 N999S/WT >Kcnma1 H444Q/WT with respect to BK current magnitude and supports the potential for N999S and D434G to cause neurobehavioral changes. The detrimental potential for Kcnma1 H444Q/WT is less clear and may require additional factors or mechanisms to support pathogenicity (i.e., other Ca 2+ conditions, cell types, or gene interactions).
These dentate granule whole-cell recordings represent an initial evaluation of the potential for each variant to affect BK currents under physiological conditions. For Kcnma1 N999S/WT , the increased BK current is not associated with higher transcript expression. Although the ratio of WT and N999S transcripts could not be individually determined from the microarray probe set, the single nucleotide mutations introduced into coding exons provide no obvious mechanism to alter the allelic expression ratios. Assuming both alleles are expressed normally, it would suggest functional changes underlie the BK current increase. However, how the properties identified from homotetramic channels ( Figure 1C-G) contribute to the increased current in neurons is unknown. The apparent voltage dependence of activation was not different between N999S, D434G, H444Q, and their respective WT control currents in neurons (data not shown). Several factors that could mitigate differences in V 1/2 recorded from homotetramers are undefined in the neurons, including the α (WT:mutant) and β subunit stoichiometry, splice variant background, and intracellular Ca 2+ . Limited data is available to consider the impact of these on heterozygous variants. Co-expression of WT and mutant (GOF) BK channel cDNAs supports the assumption that heterotetramers are the predominant channel type produced by 1:1 transcript ratios in Xenopus oocytes (Geng et al., 2021). A few studies have shown that N999S and D434G confer similar ΔV 1/2 onto different splice variants ( Figure 1C-G; Li et al., 2018;Moldenhauer et al., 2020a;Wang et al., 2009) and maintain left-shifted V 1/2 values compared to WT in the presence of the β4 subunit (Berkefeld and Fakler, 2013;Li et al., 2018;Wang et al., 2009). This data is not available for H444Q, which produced smaller effects. Yet even with D434G, less of a difference is found ±β4 above 10 μM Ca 2+ (Wang et al., 2009), which could be significant in granule neurons given the widespread abundance of β4.

N999S and D434G increase intrinsic neuronal excitability
Intrinsic excitability was next assessed in dentate granule neurons as an independent validation for neuronal pathogenicity. Both GOF and LOF BK channel mutations have the ability to alter neuronal activity in either direction, depending on the context (Bailey et al., 2019;Brenner et al., 2005;Montgomery and Meredith, 2012;Gu et al., 2007;Sausbier et al., 2004). Dentate granule cell inputoutput firing relationships were assessed in current-clamp mode ( Figure 3). Firing rates increased with current injection in each Kcnma1 WT/WT littermate control dataset, reaching a peak of ~40 Hz between 240 and 260 pA and then decreasing with higher current injections ( Figure 3A-B). Kcnma1 N999S/WT firing was greater than Kcnma1 WT/WT littermate neurons in several key places. First, across the whole current injection range, firing was significantly increased in the middle portion (160-240 pA), ranging from 25% to 30% higher than WT (Figure 3Ai, Bi). After reaching the maximum, the firing still decreased instead of remaining higher through the full range of current injections. In addition, the initial slope of firing (0-160 pA) was greater in Kcnma1 N999S/WT neurons (0.22±0.01 Hz/pA) compared to Kcnma1 WT/WT (0.18±0.01 Hz/pA, Figure 3Ci). Lastly, the maximal firing was 9.6±1.8 Hz (125%) higher in Kcnma1 N999S/ WT neurons versus Kcnma1 WT/WT (Figure 3Di). Taken together, Kcnma1 N999S/WT neurons respond to stimulation with higher firing and a shift in the input-output relationship.
Increased firing was also observed in Kcnma1 D434G/WT neurons, but the shape of the input-output alteration was different than that observed in Kcnma1 N999S/WT . Firing was 18-67% greater than WT controls at higher current injections only, from 260 to 400 pA (Figure 3Aii,Bii). Despite the increase at the higher end of the range, Kcnma1 D434G/WT firing still decreased after reaching a maximum, while remaining higher than Kcnma1 WT/WT . The initial firing rate slope was not different from Kcnma1 WT/ WT (Figure 3Cii). However, the maximal firing rate was 5.5±2.2 Hz greater (113%) for Kcnma1 D434G/ WT compared to Kcnma1 WT/WT (Figure 3Dii). This increase was shifted to higher current injections and occurred over a wider range of voltages than that observed for Kcnma1 N999S/WT .

Bi
Bii Biii Kcnma1 D434G/D434G neurons, which had the highest BK current levels, showed further differences from Kcnma1 D434G/WT . Firing was increased 22-47% in the early and middle of the current injection range, from 40 to 220 pA (Figure 3Aii, Bii). Both the initial slope (Kcnma1 D434G/D434G 0.25±0.01 Hz/pA versus 0.19±0.01 Hz/pA for Kcnma1 WT/WT ) and the maximal firing rate were greater (117%, Figure 3Cii, Dii). Yet Kcnma1 D434G/D434G firing was qualitatively more similar to Kcnma1 N999S/WT , despite the finding that Kcnma1 D434G/D434G BK current levels were almost twice as much as those recorded from Kcnma1 N999S/WT . No significant differences in firing frequency, slope for the initial firing rate gain, or maximal firing rate were observed in Kcnma1 H444Q/WT or Kcnma1 H444Q/H444Q neurons compared to WT littermates (Figure 3Aiii-Diii). The lack of significant alteration in excitability was congruent with the absence of change in BK current levels in Kcnma1 H444Q/WT neurons.

Ci Cii Ciii
We conclude that both the N999S and D434G GOF variants have pathogenic potential through their ability to increase BK currents and action potential firing. The LOF H444Q variant does not substantiate the same pathogenic potential under these conditions. Mechanistically, despite the grossly similar BK current levels between Kcnma1 D434G/WT and Kcnma1 N999S/WT neurons, the nonidentical input-output curves suggest a more complex relationship between BK channel properties and neuronal excitability in dentate granule neurons. Hints about the basis for these differences may be revealed by comparison of additional membrane parameters such as passive membrane properties and action potential waveforms. However, no differences in resting membrane potential or input resistance were observed between Kcnma1 N999S/WT , Kcnma1 D434G/WT , or Kcnma1 D434G/D434G and WT controls ( Figure 3-figure supplement 1). With respect to action potentials, modulation of repolarization and the afterhyperpolarization (AHP) in repetitive firing occurs with both BK channel inhibition (slower repolarization and reduced AHP amplitude) and activation (faster repolarization and increased AHP amplitude) (Dong et al., 2021;Gu et al., 2007;Montgomery and Meredith, 2012;Shao et al., 1999). Analysis of waveforms from the 200 pA step corroborate BK channels regulate multiple phases of the action potential, but suggested that the basis for increased firing in Kcnma1 N999S/WT and Kcnma1 D434G/D434G neurons was a faster AHP decay rate ( Figure 3-figure supplement 2), which would facilitate more rapid initiation of the next action potential. Since Kcnma1 D434G/WT neurons do not show increased firing at 200 pA, no differences would be expected in parameters related to setting firing frequency. Accordingly, no significant difference in AHP decay was found in Kcnma1 D434G/WT versus control waveforms.

N999S and D434G reduce seizure thresholds in mice
Neuronal hyperexcitability is coincident with establishment of an epileptic network, and about half of all individuals with KCNMA1 channelopathy, including those with N999S, D434G, and H444Q variants, report a history of seizures or epilepsy (Bailey et al., 2019;Miller et al., 2021). Individuals harboring the D434G variant primarily have absence seizures, if present (Du et al., 2005). Dentate gyrus hyperexcitability can both contribute to, and result from, epileptiform activity (Dengler et al., 2017;Krook-Magnuson et al., 2015;Mehranfard et al., 2015;Scharfman, 2019). In β4 -/mice, increased granule neuron firing is found in the setting of hippocampal epileptiform discharges, nonconvulsive seizures, and lower chemoconvulsant-induced seizure thresholds (Brenner et al., 2005;Whitmire et al., 2017). We hypothesized that Kcnma1 N999S/WT and Kcnma1 D434G/WT mice would show increased number, duration, or severity of seizure events compared to WT controls. However, since half of those harboring LOF variants also report seizures (Liang et al., 2019;Miller et al., 2021), including the H444Q and individuals with putative truncation alleles, Kcnma1 H444Q/WT and Kcnma1 -/mice were assessed in parallel. No seizures have been previously reported in two established Kcnma1 -/mouse models (Bailey et al., 2019; ALM unpublished data), but spontaneous epilepsy was reported in a Kcnma1-exon4 frameshift mouse line (Yao et al., 2021).
Behavioral assessments and EEGs were made from transgenic and WT littermates for indications of seizure. No spontaneous twitching/jumping/convulsions, rigidity/immobility, anorexia/dehydration, or premature mortality were observed from transgenic (or control) mice in the home cage environment. After dural electrode implantation, 24 hr baseline EEGs were recorded. No interictal epileptiform discharges, spontaneous seizures, or other abnormalities (e.g., slowing) were observed in transgenic or control mice during baselines. The absence of spontaneous events was not surprising given that half of affected individuals do not report epilepsy, and among those that do, there is a wide range in frequency (isolated to daily), semiology, and age of onset (Bailey et al., 2019;Miller et al., 2021). However, this presents challenges to evaluating spontaneous EEG events in mouse models, especially those that could be occurring in deeper brain regions similar to β4 -/mice. The presence of EEG abnormalities could be more comprehensively assessed with longer monitoring, depth electrodes, or interrogation of additional ages and strain backgrounds (Löscher et al., 2017), which were beyond the capability of the present study.
Kcnma1 N999S/WT mice developed PTZ-induced seizures that were distinguishable from Kcnma1 WT/ WT littermates in several parameters. Behaviorally, most Kcnma1 N999S/WT mice displayed tonic-clonic activity (9/13 mice modified Racine 3 or 4), with two reaching status epilepticus (2/13 mice; modified Racine 5). The latency to first seizure after PTZ injection was reduced to 75±15 s, compared to WT littermates (294±99 s; Figure 4Ai,C-D). EEG power, an estimation of seizure severity, showed a broader range with Kcnma1 N999S/WT mice, although the differences were not significant (Figure 4Bi,C-D). Interestingly despite these observations, mice exhibiting electrographic seizures did not look strikingly behaviorally different from control mice. One reason may be the movement suppression that developed in Kcnma1 N999S/WT mice after PTZ injection, quantified by EMG.   Figure 4-videos 1-4.
The online version of this article includes the following video and source data for figure 4: Source data 1. Data file for Figure 4A, B. WT mice had infrequent bouts of sustained quiescent EMG activity, with average lengths of 45±7 s (n=16). However, the inactive bouts were longer for Kcnma1 N999S/WT mice (311±126, n=10, p<0.0001, Mann-Whitney test) and were visually apparent (Figure 4-video 4-2). The movement suppression exhibited by Kcnma1 N999S/WT mice under PTZ does not have a correlate in individuals harboring N999S variants, although a few report absence seizures among other types (Miller et al., 2021). Since no spontaneous EEG + /EMGevents were observed in the baseline EEG recording period of these mice, it remains to be determined whether the PTZ-elicited movement suppression is related to an absencelike seizure manifestation. Within the D434G family, there is an intermediate penetrance for epilepsy (56%), the most frequent diagnosis being absence (Du et al., 2005;Miller et al., 2021). Like N999S, Kcnma1 D434G/WT mice also showed a reduced latency to first seizure (101±27 s) compared to Kcnma1 WT/WT mice (209±35 s; Figure 4Aii,C-D). However, this reduction was not as large as the difference between Kcnma1 N999S/WT mice and their respective controls. Total EEG power from Kcnma1 D434G/WT mice was not different from WT controls ( Figure 4Bii). Therefore, the D434G variant also increased the propensity for seizure in the transgenic model, consistent with its ability to alter neuronal excitability, but was less severe than the N999S variant. The phenotype assessed here for Kcnma1 D434G/WT is also less severe than reported in a knock-in mouse model with the D434G mutation introduced in the context of a Cre/lox cassette. Those mice showed spontaneous spike-wave discharges in both the heterozygous and homozygous configuration with complete penetrance (Dong et al., 2021), a phenotype that appears more severe than reported in the D434G pedigree, in which only half experience seizures (Du et al., 2005).
Although no patients have a homozygous D434G genotype, a limited number of Kcnma1 D434G/ D434G mice were available for EEG analysis. We tested whether Kcnma1 D434G/D434G mice, producing only mutant BK channel homotetramers, had a more severe phenotype. These mice showed a trend toward the shortest latencies to seizure, with thresholds comparable to the lowest among the Kcnma1 D434G/WT cohort (48, 58, and 69 s). Kcnma1 D434G/D434G mice had EEG power overlapping with WT and heterozygous littermates (2172, 4237, and 3627). While not conclusive and requiring substantiation in a larger cohort, the Kcnma1 D434G/D434G seizure data raise the possibility of a gene dosage effect with D434G that qualitatively differs from an independently generated D434G mouse model (Dong et al., 2021).
For LOF transgenics, changes in seizure threshold in both directions were considered. Approximately the same proportion of individuals with LOF variants report seizure as those with GOF variants (Miller et al., 2021), a finding validated in one Kcnma1 -/mouse model (Yao et al., 2021). Yet paradoxically, acute inhibition of BK channels has anti-seizure effects in other rodent models (Dong et al., 2021;Kuebler et al., 2001;Sheehan et al., 2009). However, no differences were observed in latency to first seizure or total EEG power in Kcnma1 H444Q/WT or Kcnma1 -/mice (Figure 4Aiii-Aiv,Biii-Biv, C-D; Figure 4-video 4). For H444Q, this data suggests the lack of change in dentate granule neuron BK currents and excitability may be consistent with other areas of the brain, producing no change in seizure propensity indicative of widespread hyperexcitability. Overall, alterations in BK current and firing detected concurrently with lowered seizure threshold suggests that the GOF variants N999S and D434G have the potential to contribute to seizure risk by changing neuronal activity in a mouse model. However, partial (H444Q) or total (Kcnma1 -/-) loss of BK channel function does not support the same potential in seizure etiology under equivalent conditions.

N999S and D434G cause paroxysmal dyskinesia in mice
One of the most recognizable symptoms in KCNMA1 channelopathy is a distinctive type of dyskinesia manifesting as sudden, brief paroxysms of axial hypotonia (PNKD3). These episodes sometimes resemble the immobility in non-narcoleptic cataplexy, but have preservation of some muscle tone that varies among individuals (Du et al., 2005;Heim et al., 2020;Keros et al., 2022;Miller et al., 2021;Wang et al., 2017;Zhang et al., 2015). Patients may slump or fall over but can often maintain position if appropriately supported, and consciousness is maintained. Normal activity is recovered  Braverman, 2019;Sanders, 2018). PNKD3 episodes are not initiated by movement or exertion (non-kinesigenic), but rather by negative and positive triggers such as strong emotion (stress and excitement), cold, fatigue, or alcohol. The events are not associated with epileptiform activity on EEG and are generally unresponsive to anti-seizure medications (Keros et al., 2022;Miller et al., 2021). PNKD3 is associated with substantial morbidity due to its high frequency, with hundreds of episodes per day. All three variants tested in this study are associated with PNKD, with 75% of individuals harboring N999S and D434G carrying the diagnosis but also observed at lower incidence with LOF variants or VUS (variant of uncertain significance) (Miller et al., 2021).
There are currently no standardized behavioral assays for either PNKD3 or non-KCNMA1-associated PNKD. In other paroxysmal dyskinesia animal models, the phenotype is usually hyperkinetic, not the hypokinetic events observed in PNKD3. For example, Ca 2+ channelopathy, Prrt2-deficient, and Pnkd mutant mice are characterized by dystonia, chorea, and tonic-clonic episodes (Fureman et al., 2002;Lee et al., 2012;Michetti et al., 2017;Tan et al., 2018;Pan et al., 2020). No spontaneous hypotonic dyskinetic motor behavior was detectable to a blinded observer in any of the transgenic lines in this study. Therefore, we sought to elicit episodes by utilizing known triggers for PNKD3. Since individuals harboring N999S and H444Q variants are mostly children without any reported alcohol exposures, a PNKD trigger specifically reported for D434G (Du et al., 2005), and no calibratable emotional responses are validated in mice, we focused on the standardizable stress experienced during physical restraint. Stress provocation is the closest stimulus to the natural triggers observed in PNKD3-affected individuals (Miller et al., 2021). Restraint stress provoked dyskinesia in most (85%) tottering mice (Fureman et al., 2002), and PNKD mutant mice also showed dyskinesia after stressful handling when placed in a beaker (Lee et al., 2012).
To test whether restraint stress would produce paroxysmal dyskinesia, mice were subjected to an acute stereotypical manual restraint protocol by an experienced handler. After restraint, mice were placed into a beaker, a novel-constrained environment proposed to enhance stress (Lee et al., 2012). Mice with PNKD-like characteristics were predicted to show restraint-triggered hypokinetic episodes. Mice were scored for abnormal movement (time immobile, circling/hyperactivity, twisting/chorea or limb-clasping, tonic-clonic movement, flattened/dystonic posturing, tremor, listing and falling) in the beaker under video observation. Stereotypical behaviors such as grooming were also recorded. WT mice from all groups showed normal exploratory behavior including sniffing, grooming, and rearing with coordinated movements.
Kcnma1 N999S/WT mice and WT littermates placed in the beaker without prior restraint did not show any dyskinetic movements or collapsing behavior (paws no longer touching the ground). There was no significant difference in the time spent immobile between these groups ( Figure 5A). Next, restrained mice were placed in the beaker. Since mice increase grooming when released from stress Shirley et al., 2008, Jones andMormède, 2000, this behavior was used as a control to indicate the presence of stress. Kcnma1 N999S/WT mice and WT littermates both showed an increase in grooming events after restraint compared to their non-restraint controls ( Figure 5B), confirming both genotypes responded to stress with an increase in stereotypical behavior.
After stress, Kcnma1 WT/WT mice had exploratory behavior and spent less than a minute immobile in the beaker (51±10 s). Although the range was wider, their time spent immobile did not differ significantly from the unrestrained baseline. In contrast, Kcnma1 N999S/WT mice were immobile for more than twice as long after stress (120±12 s) ( Figure 5A, Figure 5-video 1). After episodes of immobility, both genotypes resumed normal exploratory behavior or grooming.
In qualitative assessment, three Kcnma1 N999S/WT mice had extended myoclonic 'hiccups' throughout the immobility that were not associated with respiratory rate. One mouse also showed listing, and three had a flattened posture. Evaluation of other dyskinetic behaviors (dystonia, chorea, clasping, etc.) in non-restraint controls and after stress revealed grossly normal movements for Kcnma1 N999S/ WT mice, with the exception of the notable immobility. In direct comparison, Kcnma1 WT/WT littermates had raised heads and less hunched postures during their briefer immobility, suggesting the maintenance of normal axial tone. Brief hiccups were observed in one WT control, at shorter duration than the Kcnma1 N999S/WT mice, and one mouse had a brief flattened posture during the first bin. Use of a fitted tube restraint, which may produce a stronger stress response, increased the ability of a blinded observer to predict genotype differences in immobility (n=11 mice, data not shown).
Six PNKD3-affected individuals harboring the N999S variant have been documented to experience nearly full resolution of immobilizing episodes with lisdexamfetamine, a prodrug of dextroamphetamine (d-amp; Keros et al., 2022). D-amp treatment is also highly effective against PNKD3 associated with another GOF KCNMA1 variant (N536H; Zhang et al., 2020). Acute daily d-amp administration reduces the number of PNKD3 episodes during the therapeutic window from >100 to 0 in some cases (Keros et al., 2022). If the stress-induced immobile state in Kcnma1 N999S/WT mice is consistent with PNKD3, administering d-amp would be predicted to reduce immobility time.
To test for rescue effects, Kcnma1 N999S/WT mice and WT littermates were injected with low dose d-amp (0.5 mg/kg), similar to PNKD3-affected patients taking lisdexamfetamine or d-amp (Keros et al., 2022;Zhang et al., 2020). Mice were then subjected to 5 min of restraint stress, followed  Representative videos for this dataset appear in Figure 5-video 5-1.
The online version of this article includes the following video, source data, and figure supplement(s) for figure 5: Source data 1. Data file for Figure 5A-F.   with observation of mobility state in the beaker assay ( Figure 5C). Experimental and separate control assays were conducted during the peak time window reported for d-amp effect from open field locomotion (30 min post-injection; Fu et al., 2021;Gould et al., 2007). As a control, d-amp administration without restraint stress does not affect immobility in either Kcnma1 N999S/WT mice or WT littermates ( Figure 5-figure supplement 1A). This corroborates that after an acute injection, the restraint stress stimulus is still required to produce immobility in Kcnma1 N999S/WT mice ( Figure 5C). Furthermore, d-amp does not increase wheel running or increase grooming in either genotype ( Figure 5-figure  supplement 1B-C), similar to prior C57BL/6J studies (Cytryn, 1980). These control data further indicate that general locomotor activity and the stress response are not markedly increased under the d-amp dosing conditions. Under the restraint stress paradigm, saline-injected Kcnma1 WT/WT mice showed normal exploratory behavior with little immobility (<10 s), with one exception of a single mouse immobilized for 121 s exhibiting a hunched posture. D-amp treatment was not different from saline control for Kcnma1 WT/WT mice ( Figure 5C). In contrast, restrained Kcnma1 N999S/WT mice produced a large increase in immobility after saline injection (>4-fold average), corroborating un-injected animals subjected to restraint stress ( Figure 5A and C). The majority of saline-injected Kcnma1 N999S/WT mice spent 1-2 min immobile and assumed a hunched posture with the head lowered.
D-amp-injected Kcnma1 N999S/WT exhibited little immobility compared to saline-injected Kcnma1 N999S/ WT mice (3-fold less). These mice had normal exploratory behavior with shorter, more frequent episodes of grooming. However, when present, the shorter immobility bouts were associated with hunched posture. After d-amp treatment, there was no statistically significant difference in immobility between Kcnma1 N999S/WT mice and WT littermates ( Figure 5C). Automated analysis of the assays by an additional blinded experimenter replicated the finding of abrogation of immobility with d-amp treatment in Kcnma1 N999S/WT mice ( Figure 5-figure supplement 1D). In conclusion, these data show that d-amp treatment of Kcnma1 N999S/WT mice rescues stress-induced immobility at therapeutically relevant doses.
Taken together, these data suggest that the presence of a stressor (restraint) produces a new behavioral state in Kcnma1 N999S/WT mice (immobility) that was not observed in the absence of the trigger or in WT littermates. If the immobility behavior resulted from stress-induced atonic or absence seizures, these events would likely have been observed during baseline EEG recordings given the number of occurrences in the 5 min beaker assay. However, seizure would not be expected to resolve with low-dose d-amp. Alternatively, if stress-induced immobility resulted from general hypoactivity or altered fear response, open field testing might show a difference in motor exploratory behavior between Kcnma1 N999S/WT and WT littermates. This was not observed ( Figure 6B). Kcnma1 N999S/WT mice were also able to achieve the same peak speed as WT littermates during voluntary wheel running ( Figure 6Aii). In addition, there is no evidence from patients for correlation of PNKD3 with increased anxiety, depression, or hypoactivity (Miller et al., 2021). We conclude that stress-induced immobility, which occurs in brief episodes that are instantaneously recovered, is responsive to d-amp, and occurs without other hyperkinetic or tonic-clonic manifestations, is consistent with the reversible triggered hypokinetic behavioral state in PNKD3-affected individuals (Heim et al., 2020;Keros et al., 2022).
To further characterize this mouse model for PNKD3, additional motor assays were conducted. Besides stress, PNKD3 episodes can be triggered by positive emotions or excitement, similar to cataplexy in patients with narcolepsy (Dauvilliers et al., 2014;Miller et al., 2021;Kelley, 2001Sun et al., 2019a. Related to the reward and arousal effects in mice, cataplexy can be provoked in narcoleptic orexin-deficient (Hcrt -/-) mice by wheel running (España et al., 2007;Mahoney et al., 2017;Novak et al., 2012). We also assessed this positive trigger to determine if voluntary wheel running could produce a PNKD-like behavior in the setting of a more complex motor task. In this assay, Kcnma1 N999S/ WT mice covered a shorter distance compared to their WT littermates (Figure 6Ai). The reduction in running distance was not due to a decrease in the maximum speed the mice could achieve, but instead occurred as a result of increased gaps in activity (Figure 6Aii-iv). These gaps could be consistent with, but not exclusively attributable to, cessation of running during a PNKD-like immobility. However, we cannot rule out the reduced distance as a result of a baseline dyskinesia or muscle weakness, since Kcnma1 N999S/WT mice also show decreased time to fall in the rotarod and hanging wire assays ( Figure 6C-D).
PNKD3 is also exhibited in individuals harboring heterozygous D434G variants and is provoked by additional triggers besides stress, such as alcohol (Du et al., 2005). However, without diagnostic standardization, it is not clear whether this constitutes a different type of PNKD episode in individuals harboring D434G compared to N999S. In the stress assay, Kcnma1 D434G/WT mice and WT littermates had similar immobility lasting 53±9 and 63±6 s, respectively ( Figure 5D). However, in homozygous Kcnma1 D434G/D434G mice, immobility time was similar to N999S heterozygotes (101±11 s). Thus, stressinduced dyskinesia is present in the D434G mouse model. It remains possible that alcohol would also be capable of triggering these episodes, but it is difficult to assess given the bi-directional motor effects of alcohol in mice (Jones and Mormède, 2000). Kcnma1 D434G/D434G mice have aspects of reduced motor coordination on the rotarod and running wheels but are not hypokinetic under basal conditions ( Figure 6-figure supplements 1 and 2).   (n=11) on day 2 (*p=0.0045) and day 7 (*p=0.0124) compared to Kcnma1 N999S/WT mice (n=12). Motor learning was observable as an improvement in fall latency times across the three trials on each day (data not shown), suggesting the overall impairment was related to motor coordination and not learning. Data are presented as individual data points with median and inter-quartile range (A-C) and mean ± SEM (D). Results for these assays with Kcnma1 D434G , Kcnma1 H444Q , and Kcnma1 -/mice appear in Figure 6-figure supplements 1 and 2. For these assays, the baseline motor coordination severity fell in the series Kcnma1 -/->Kcnma1 D434G/D434G > Kcnma1 N999S/WT >Kcnma1 H444Q/H444Q .
The online version of this article includes the following source data and figure supplement(s) for figure 6: Source data 1. Data file for Figure 6Ai, Aiv, B-DD.    In contrast, homozygous LOF manipulations showed a different directionality in stress-triggered dyskinetic behavior, with less immobility after restraint than either WT or heterozygous littermates. No phenotypic differences were detected in heterozygous mice of either line. However, immobility in Kcnma1 H444Q/H444Q mice was reduced to 16±6 s ( Figure 5E), and in Kcnma1 -/mice, it was reduced to 27±7 s ( Figure 5F). Three Kcnma1 -/mice also showed hyperactive circling and rapid limb movements notable to the blinded observer, and one had notable tremor during the brief non-active periods. Kcnma1 H444Q/H444Q and Kcnma1 -/mice had reduced latencies to fall from the hanging wire under basal conditions, but only Kcnma1 -/mice are profoundly impaired on the rotarod and running wheels due to ataxia (Figure 6-figure supplements 1 and 2). The reduced motor performance in Kcnma1 H444Q/H444Q and Kcnma1 -/mice stands in contrast to the reduction in immobility observed after stress ( Figure 5E-F). These data reveal that the H444Q variant and KCNMA1 null genotypes are not associated with PNKD immobility under the same triggers that provoke GOF variants. The results raise the possibility that stress-induced dyskinesia manifestation is influenced by mutation type, with GOF producing hypokinetic and LOF producing hyperkinetic responses.

Discussion
We have characterized the channel properties, neuronal activity, neurobehavioral phenotypes, and relative severity of three KCNMA1-linked channelopathy variants under equivalent conditions. Pathogenic potential was established using four criteria (MacArthur et al., 2014): (1) low variant frequency in the human population, classifying as a mutation (Miller et al., 2021), (2) variant alters BK channel gating properties, (3) variant alters neuronal BK currents and firing, since the channelopathy is a neurological disorder, and (4) variant produces phenotypes similar to the central patient diagnosesseizure susceptibility and PNKD. The findings support the conclusion that KCNMA1-linked channelopathy, although symptomatically heterogenous and comprised predominantly of de novo variants, has the potential to be categorized as a monogenic disorder. The results substantiate hyperexcitability, increased seizure propensity, and PNKD as collective phenotypes replicated in two hypermorphic GOF KCNMA1 alleles. Moreover, our data for N999S and D434G corroborate mounting evidence, both in patients (Du et al., 2005;Keros et al., 2022;Miller et al., 2021;Zhang et al., 2020) and in animal models (Dong et al., 2021;Kratschmer et al., 2021), that PNKD can be considered the most consistent symptom for KCNMA1 GOF channelopathy.
N999S produced the strongest GOF effect on BK channel properties under these study conditions. Although N999S is the most commonly reported KCNMA1 variant, direct evidence that it caused channelopathy was lacking because it arose de novo in all known cases (Keros et al., 2022;Miller et al., 2021). In mice, our data validate its dominant inheritance and pathogenic potential as a GOF mutation able to increase BK current and neuronal activity in the heterozygous configuration in mice. Neurobehavioral validation further identified increased PTZ-induced seizure propensity and stress-triggered dyskinesia episodes resembling PNKD-like immobility, broadly consistent with the phenotypic occurrence in patients. The lethality of homozygous Kcnma1 N999S/N999S (and hemizygous Kcnma1 N999S/Δ ) genotypes, which have not been found in any patient, underscore the severity of this variant. It is not known which cell type the lethality stems from, but N999S channels can produce both increased peak action potential-evoked and subthreshold BK current (Moldenhauer et al., 2020b). Thus it is possible that N999S could increase firing in some cells, exemplified by the effect in dentate granule neurons, or prevent firing, depending on the cellular context. D434G, a less severe GOF mutation than N999S at the BK channel level, had dominant inheritance for a subset of traits in our model, partially paralleling the familial pedigree (Du et al., 2005). The increased BK current, excitability, and PTZ-induced seizure propensity in the heterozygous configuration validated D434G pathogenicity. However, homozygosity was required to produce the PNKD-like immobility attacks, ranking D434G less pathogenic than N999S. While Kcnma1 D434G/D434G mice would produce obligate homotetrameric mutant BK channels, additional work is needed to understand whether the decreased severity in Kcnma1 D434G/WT mice is indicative of heterotetrameric (WT:D434G) BK channel formation. Interestingly, three other D434G models have been reported, with some phenotypic variation. BAC-loxP-D434G mice exhibited GTC seizures in the absence of motor dysfunction . Another Cre/lox-D434G mouse line was comparatively more severe with complete penetrance of absence seizures and dyskinesia in the heterozygous configuration (Dong et al., 2021). This variability, observed in both mouse models and incomplete penetrance in patients, raises the possibility that additional genetic or environmental factors can influence symptomatic severity. Nevertheless, an analogous mutation in flies also alters neuronal activity and baseline motor behavior (Kratschmer et al., 2021).
The LOF variant, H444Q, demonstrated limited pathogenicity by decreasing BK channel activity but was not validated as a hypomorphic or haploinsufficient allele in neurons or mice. Homozygous Kcnma1 H444Q/H444Q mice showed a neurobehavioral phenotype distinct from PNKD: stress-induced hyperkinetic motor responses similar to Kcnma1 -/null mice. Given multiple genetic findings and symptomatic ambiguity in the patient carrying this variant, the different dyskinetic responses compared to the two GOF models may suggest a basis to improve the diagnostic investigations for this and other KCNMA1 variants classified as LOF or VUS. At present, there are no patients with homozygous KCNMA1 alleles validated as functionally null for channel activity (Miller et al., 2021), but the ataxia, tremor, decreased strength, and hyperactivity in Kcnma1 -/mice (Imlach et al., 2008;Meredith et al., 2004;Meredith et al., 2006;Sausbier et al., 2004;Typlt et al., 2013;Wang et al., 2020) are symptoms observed at lower incidence among patients. Lastly, our experimental conditions failed to corroborate the influence of LOF alleles on seizure propensity predicted from several animal studies Kuebler et al., 2001;Pacheco Otalora et al., 2008;Sheehan et al., 2009;Shruti et al., 2008;Yao et al., 2021). KCNMA1 LOF channelopathy has also been proposed to carry a broader set of non-overlapping features associated with a subset of de novo LOF variants, referred to as Liang-Wang syndrome (Liang et al., 2019). However, none of the observable patient correlates were present in the LOF H444Q model studied here. H444Q mice also had little overlap with Ryegrass Staggers, a toxicity syndrome of livestock involving BK channel inhibition that is phenotypically similar to Kcnma1 -/mice (Imlach et al., 2008).
Genotype-phenotype relationships are important for understanding KCNMA1 channelopathy disease mechanisms as well as potential therapeutics. The allelic series established from N999S, D434G, and H444Q BK channels in vitro is outwardly congruent with the relative severity in mice. Such allelic series have been pivotal in understanding other complex channelopathies, especially those delineating distinct disorders within the same gene association (Noebels, 2003;Pietrobon, 2005;Zwingman et al., 2001). However, it is not yet clear whether all KCNMA1 variants discovered in the setting of neurological diagnoses carry the same pathogenic potential. Because this KCNMA1 allelic series was derived from a limited set of conditions, designations of phenotypic severity could be further influenced by additional factors.
At the DNA level, it is likely that all three variant-containing alleles are expressed because the homozygous phenotypes are dissimilar to Kcnma1 -/-: Kcnma1 N999S/N999S and Kcnma1 D434G/D434G have unique phenotypes not found in Kcnma1 -/-(lethality and stress-induced immobility, respectively), and Kcnma1 H444Q/H444Q had fewer and less severe phenotypes than Kcnma1 -/-(e.g., rotarod). However, the phenotypic severity for the human variants could be mitigated by their context within the mouse gene, which was not humanized through additional rounds of gene editing. Mouse and human BK channels differ at eight constitutive coding residues and have minor differences in BK current properties in heterologous cells (Lai, 2015). D434G produces a larger GOF effect on the G-V relationship in the context of a human BK channel compared to mouse (Wang et al., 2009). Similarly, as the least potent variant, it is possible that H444Q has a further reduced effect on BK channel properties in mouse, potentially contributing to the lack of BK current differences.
At the channel level, unresolved questions concerning functional mechanisms and subunit composition illustrate the influence of additional factors besides variant genotype on the phenotypic severity. For example, BK current levels were relatively similar between Kcnma1 N999S/WT and Kcnma1 D434G/WT in one type of neuron (dentate granule), yet these two heterozygous genotypes exhibited differences in excitability. Instead, homozygous Kcnma1 D434G/D434G firing curves were more comparable to Kcnma1 N999S/WT . Underlying this, the ratio of expression from WT and mutant alleles, stoichiometry and properties of heterotetrameric channels, alternative splice variation, and the composition of α:β:Ca V macrocomplexes in the loci responsible for neuropathology in heterozygous transgenic mice all still remain to be resolved. Additional experiments to probe the voltage and Ca 2+ -dependent bases for N999S and D434G gating defects (Diez-Sampedro et al., 2006;Du et al., 2005;Li et al., 2018;Moldenhauer et al., 2020b;Yang et al., 2010), and the manifestation in the presence of the highly expressed β4 subunit (Wang et al., 2009;Berkefeld and Fakler, 2013;Wang et al., 2016), may also further explain these relative differences in BK currents and pathogenicity. Interestingly, no KCNMB4 variants associated with seizure have been described yet, even as the number of seizure-associated BKα variants has increased (Miller et al., 2021). This could suggest that loss of β4 regulation would have more severe consequences.
At the neurobehavioral level, increased dentate granule cell excitability may contribute directly or indirectly to the reduced seizure thresholds, but other areas of the brain are additionally involved in the generalized PTZ-evoked seizures. The BK channel inhibitor paxilline can block chemoconvulsantinduced seizures, associated with changes in cortical excitability (Sheehan et al., 2009;Shruti et al., 2008). These data suggest additional neurons to investigate for links between GOF BK channels and hyperexcitability. It is also not clear yet how PNKD-like symptomology is produced. BK channels regulate excitability in several motor-associated regions including the cerebellum, striatum, neuromuscular junction, and skeletal muscle (Abrahao et al., 2017;Goldberg and Wilson, 2005;Sausbier et al., 2005;Tricarico et al., 1997;Vatanpour and Harvey, 1995;Wang et al., 2020). Cre/lox-D434G and PRRT2 mice show changes in cerebellar excitability and morphology (Calame et al., 2020;Dong et al., 2021) but do not show hypokinetic PNKD described in this study. In other PNKD animal models, as in patients, the brain mechanisms are not well understood. Some PNKDs respond to antiepileptic medications, a few respond to deep-brain stimulation, but most necessitate trigger avoidance (Manso-Calderón, 2019). In PNKD3, stimulants (lisdexamfetamine and dextroamphetamine) are highly effective in reducing attacks described as both dystonic and hypotonic (Keros et al., 2022;Zhang et al., 2020), but neither drug has a direct effect on BK WT channel activity (Figure 1-figure  supplement 2 and Zhang et al., 2020) or BK N999S (Figure 1-figure supplement 2) in heterologous cells, leaving the target for their actions on PNKD3 an open question. The significant reduction in stress-induced immobility with d-amp treatment in Kcnma1 N999S/WT mice substantiates the utility of this model in future studies dissecting the cell and circuit basis for the PNKD3. Clinical observations and d-amp responsiveness in patients predict a central neurological dysfunction in producing the debilitating hypokinetic state (Keros et al., 2022;Zhang et al., 2020), but whether other factors such as altered neuromuscular transmission or episodic muscle hypotonia make some contribution during attacks remains to be systematically tested.
Changes in BK channel function and/or KCNMA1 expression are associated with a growing number of neurodevelopmental disorders including epilepsy, dyskinesia, autism, Angelman's syndrome, Fragile X syndrome, and brain and skeletal malformations (Cheng et al., 2021;Deng and Klyachko, 2016;Du et al., 2020;Kessi et al., 2020;Kshatri et al., 2020;Laumonnier et al., 2006;Liang et al., 2019;Miller et al., 2021;N'Gouemo, 2014;Sun et al., 2019a;Sun et al., 2019b). Neuropathology in these disorders is associated with changes in BK channel activity in both directions. Yet it has been challenging to distill KCNMA1-linked channelopathy into a cohesive GOF versus LOF symptomology because the existing patient data lack genetic pedigrees and diagnostic cross-comparability. Looking ahead, the phenotypic penetrance and heterogeneity investigated here validate only a few of the 40+ patient-associated KCNMA1 variants, but it will not be possible to make transgenic models for every case. There is less symptomatic consistency among non-GOF alleles (LOF or VUS), identifying this as a potentially fruitful area for future investigations.

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Key resources
Macroscopic currents were recorded with a Multiclamp 700B amplifier, and signals were filtered at 10 kHz and digitized at 50 kHz using Digidata1440A and pCLAMP v10 software (Molecular Devices, Sunnyvale, CA). BK currents were activated with a voltage protocol stepped from a holding potential of −100 mV stepping to +250 mV with +10 mV increments for 30 ms and back to −100 mV for 15 ms to generate tail currents. G-V curves were generated from the tail currents 150-200 µs after the peak normalized to the maximum conductance (G/G max ) and plotted against the activating voltage step (V). V 1/2 values were calculated from a Boltzmann fit of the G-V curves (Prism v9 GraphPad Software, San Diego, CA). Leak currents were compensated using a P/5 protocol with a subsweep holding potential of −120 mV.
Activation time constants were obtained from the same patches in Figure 1. τ act was obtained by fitting the rising phase of the outward K + current to single exponential function. For the deactivation kinetics, BK currents were elicited by +200 mV voltage command for 20 ms from a holding potential of −100 mV followed by 15 ms voltage steps from -200 to -10 mV with +10 mV increments. Deactivation time constants were obtained by fitting tail currents with single exponential functions. Leak currents were compensated using a P/5 protocol with a subsweep holding potential of −120 mV.
For experiments with dextroamphetamine and lisdexamfetamine, BK currents were recorded in inside-out patches in physiological K + and 10 μM intracellular Ca 2+ as described in Moldenhauer et al., 2020a. In voltage-clamp mode, patches were held at -150 mV, stepped from -150 to +150 mV for 30 ms (10 mV increments), and stepped back to -150 mV. Lisdexamfetamine dimesylate (catalog L-026, Supelco Millipore-Sigma) and dextroamphetamine (catalog 1180004, Millipore Sigma) were applied at 155 ng/ml, and paxilline (#2006; Tocris, Bristol, UK) was applied at 100 ng/ml. Current levels were assessed at baseline and 5 min after drug application and were normalized to control current levels for each patch.
Kcnma1 -/-(Slo KO) mice were maintained on a C57BL/6J background (>N12 generation). Littermates of each genotype were produced from heterozygous Kcnma1 +/-× Kcnma1 +/breeding pairs, as described previously (Meredith et al., 2004) using primer sequences described in 'Genotyping of Kcnma1 N999S , Kcnma1 D434G , Kcnma1 H444Q , and Kcnma1 −/− mouse lines' section. For all lines, male and female mice were separated by sex and group housed on a 12 hr light/12 hr dark cycle. Chow and water were provided ad libitum. Kcnma1 N999S/WT breeders were fed with high fat chow supplement to enhance breeding. All experiments were conducted in accordance with the University of Maryland School of Medicine Animal Care and Use Guidelines and approved by the Institutional Animal Care and Use Committee (Protocol #1120011). Sex-matched WT and transgenic littermates of both sexes were used for experimental procedures at the indicated ages. Experimenters were blinded to animal genotype at data collection and analysis.

Gene expression
Unilateral hippocampus and the medial portion of cerebellum were extracted from 4-month-old mice, and directly put them in 1.0 mm diameter zirconium beads with 750 µl of trizol for bead homogenization. RNA was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. To assess the RNA quality, RNA was quantified via RNA-40 nanodrop and OD 260/280 ratio of all samples were between 1.94 and 2.05. Integrity of RNA was examined via Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA integrity number of all samples were >9. For transcriptome analysis, mouse Clariom D Assay (Applied Biosystems, Waltham, MA) was used following the manufacturer's instruction. RNA extraction and array processing were done at the Genomics Core Facility, University of Maryland, Baltimore, MD. The raw microarray profiling data was preprocessed and quartile-normalized using the Transcriptome Analysis Console Software (version 4.0.1) (accessed on December 1, 2021). All samples passed array quality control evaluation. Data normalization, differential expression, and hierarchical clustering analysis was performed with default parameters by an experimenter blinded to genotype and brain region.
In current-clamp recordings, the resting membrane potential was measured without any current input after a whole-cell mode was made. If the initial resting membrane potential was more depolarized than -70 mV or a series resistance was larger than 20 MΩ, the recording was not proceeded. The membrane potential was maintained at around -80 mV by injecting positive currents. Firing frequency was obtained from running a single protocol containing 1 s step current injections (0 to +400 pA, 20 pA increment, 10 s ISI). Bridge balance was used. The input resistance (R i ) was measured with a linear regression of voltage changes from 400 ms hyperpolarizing current injections (-40 to -10 pA in 10 pA increments). The membrane time constant was calculated from the averaged traces to 20 consecutive hyperpolarizing current pulses (−20 pA; 400 ms) with a single exponential function (Lopez et al., 2012). If the series resistance (R s ) or membrane capacitance (C m ) was changed more than 20% over the recording, the cell was not further analyzed.
In voltage-clamp recordings, R s was compensated at least 60%. BK currents were measured by subtracting currents with 10 μM paxilline from the total current in the presence of 1 μM tetrodotoxin (TTX) and 2 mM 4-aminopyridine (Montgomery and Meredith, 2012). Cells were held at −90 mV, 150 ms voltage step of -100 to +30 mV in 10 mV increments was applied and stepped back to -90 mV for 130 ms. Three current traces were averaged for analysis, and leak currents were subtracted using the P/4 method with a subsweep holding potential of −90 mV. Paxilline was applied to the slice using a local perfusion pencil for at least 10 min prior to the second recording. BK current levels were obtained from the peak and normalized to cell capacitance. No paxilline-sensitive current was present in Kcnma1 −/− dentate granule neurons (n=2).

Action potential waveform analysis
Action potential amplitude was defined as the difference between the peak and threshold. Half width (t 1/2 ) was the width of action potential at 50% of the peak amplitude from the action potential threshold. The amplitude of fAHP was defined as the voltage change from the action potential threshold to the most negative voltage (AHP anti-peak) after repolarization. The fAHP decay was measured as the depolarization rate from the AHP anti-peak over the first 3 ms (ΔV/3 ms). Action potential thresholds, defined as the membrane potential where the first derivative reached 10 mV/ms, were not different between genotypes for any mouse line (data not shown).

EEG and EMG recordings
Behavioral observations, surgeries, EEG and EMG recordings and data analyses were performed blinded to experimental condition and genotype. After daily monitoring of behavioral signs of seizures, 2-to 4-month-old mice were implanted with dural electrodes, with or without EMG lead implantation at the dorsal clavotrapezious neck muscles behind the base of the skull Pinnacle Technology 4 channel EEG system, Lawrence, KS (Iffland et al., 2020). Following a minimum of 72 hr of recovery period, video-EEG recordings were obtained using Pinnacle Technology Sirenia Acquisition software for 24 hr at a sampling rate of 2 kHz. Mice were visually monitored during seizures and behavioral responses were scored using modified Racine criteria: (1) raised tail and/or abnormal posturing; (2) myoclonic movement of a limb, favoring one side; (3) brief tonic-clonic convulsive episodes (approx. 1-5 s); (4) tonic-clonic seizures associated with rearing or jumping; and (5) status epilepticus (Lüttjohann et al., 2009;Van Erum et al., 2019). Continuous EEGs were manually reviewed for interictal epileptiform discharges and/or spontaneous seizures. Interictal epileptiform discharges were defined as discrete and sharply contoured discharges (e.g., spike and wave). Seizures were defined as at least 10 s of sharply contoured and rhythmic bursts of activity.
Seizure thresholds were defined in response to a single chemoconvulsant challenge in naïve animals. A baseline 15 min video-EEG recording was obtained, followed by injection of 40 mg/kg of PTZ (Sigma, Cat. #P6500, 10 mg/ml stock in sterile saline) and an additional 30 min recording after injection. Thirty minutes post-PTZ injection, the experiment was terminated, and mice were euthanized by CO 2 asphyxiation and thoracotomy. Video-EEG and EMG data were analyzed using Sirenia Seizure Pro (Pinnacle Technology, Lawrence, KS). Experimenters were blinded to experimental group during PTZ-induced seizure, observation, and data analysis. Seizures were defined as 10 continuous seconds of sharply contoured and rhythmic discharges with a clear onset, offset, and evolution. Seizure threshold was defined as the latency to first seizure after PTZ injection. Total EEG power was reported as the sum of all frequency bands. EEG traces were examined visually for significant artifacts, and EEGs resulting in anomalous power data were excluded from the analysis, defined as EEGs with high amplitude movement artifacts (>16,000 µV 2 /Hz) or low amplitude signals (<750 µV 2 /Hz). EMG data were analyzed by manual review and the longest durations of attenuated EMG activity were scored. Attenuated EMG activity was defined as at least 1 s of EMG activity that was lower in amplitude than pre-PTZ injection baseline.

Stress-induced dyskinesia assays
Two-to 3-month-old mice were used in all assays for N999S and D434G cohorts. Two-to 8-month-old mice were used for Kcnma1 −/− cohorts due to breeding difficulties (Meredith et al., 2004). For acute stress-induced dyskinesia evaluation, the total restraint time was 5 min. Mice were restrained for 2.5 min by hand, clasping the dorsal cervical aspect between the index finger and thumb and the tail with the pinky finger, with the mouse dorsal side flat against the palm in a vertical upright position. Afterward, the tail was released, leaving only the upper body restrained for 2.5 min. In separate experiments, dextroamphetamine (d-amp) was administered via intraperitoneal (IP) injection at 0.5 mg/kg, or an equivalent volume of saline, to a final volume of 5 ml/kg. Syringes were prepared by a second blinded experimenter. Thirty minutes after injection, mice were restrained in a plexiglass tube (MH-100, IBI Scientific, Dubuque, IA) for 5 min.
After restraint, mice were placed in a transparent 1000 ml beaker under video recording for 5 min. Behavioral parameters were modified from stereotypic behavioral scoring (Kelley, 2001) and prior dyskinesia mouse models (Khan et al., 2004;Khan and Jinnah, 2002;Sebastianutto et al., 2016;Shirley et al., 2008) and were manually scored from side-view videos totaled for grooming time, or the number of occurrences of rearing, sniffing, circling/hyperactivity, twisting/choreiform movement, tonic-clonic movement, flattened/dystonic posturing, tremoring, listing, and falling. Immobility/ behavioral arrest was defined as lack of positional and translational movement from the body, limbs, and head, excluding involuntary movement from respiration. Episodes were cumulatively timed to obtain the total immobility in 5 min. Videos from d-amp-injected mice were additionally subjected to automated analysis (EthoVision XT 11.5, Noldus) by a third blinded experimenter. Movement tracking was calibrated from side-view video to beaker dimensions: rim = 11.110 cm, base = 10.795 cm, and height = 15.875 cm. Immobility time was obtained from the center point movement parameter with automated body detection. Mice were considered mobile with center point velocities exceeding 0.151 cm/s, and immobility time was obtained from intervals where the center point velocity was below 0.050 cm/s.

Open field activity
Mice were acclimated in the testing room 1 hr prior to assays. Each mouse was placed in the open arena (70 × 30 × 25 cm 3 , Cover et al., 2019) for 15 min. Mouse movement and total distance was analyzed in EthoVision XT (Noldus).

Wheel running activity
Mice were placed in housing cages with running wheels (Coulbourn Instruments) on a standard 12:12 hr light-dark cycle for 48 hr with ad libitum access to food and water. Wheel activity was measured via magnetic switches and recorded using ClockLab software (Actimetrics). Individual mouse wheel rotation counts were then quantified in 1 min bins in ClockLab software running in Matlab v6.1 (Mathworks). The following parameters were calculated for the 12 hr dark phase as average measurements: speed, maximum speed, number of activity gaps (defined as consecutive 1 min bins registering 0 rpm), activity gap duration, and maximum activity gap duration. All parameters were calculated by a custom python script (code provided as a source data file).
In separate mice, baseline wheel activity recording was collected (30 min), followed by 0.5 mg/kg d-amp or saline injections as described in the previous section. Thirty minutes after injection, each mouse's post-treatment activity count (30 min) was normalized to the baseline.

Rotarod
Motor coordination was tested by rotarod over 7 days of trials on an accelerating rod. Mice were acclimated to the testing environment in their normal housing cages for 1 hr prior to testing. Mice were trialed three times a day for 7 consecutive days under video capture. Body weight was measured on days 1 and 7. After placement on the rotarod (IITC Life Science Inc, Rat Mouse Rotarod), mice were acclimated on the apparatus for 30 s prior to first trial. During the acclimation, mice were allowed to fall off up to two times. Rod acceleration was 4-40 rpm over 5 min for each trial. Mice were given a 2 min inter-trial interval. 'Fall' was called when the mice fell off from the rod or made a 360 degree revolution around the rod. Three trials per day were averaged for each mouse.
Hypotonia and PNKD-like paroxysms would be expected to produce extremely short latencies to fall, since a single major loss of tone would be catastrophic for coordination. Alternately, baseline coordination could be impaired in the absence of immobility. Among the individual mice, there was no explicit evidence for individual trials with extremely short fall latencies that would be consistent with the triggering of sudden hypotonic events. However, partial loss of tone might be compensated for by the high level of attention induced in this assay. Compared to the voluntary running wheel activity, successful navigation of the rod's surface and rotation requires a higher degree of attention to motor coordination than the wider home cage wheel. Thus, mice fall off the rotarod at lower speeds than the maximum speeds achieved on the voluntary running wheel.

Hanging wire
Acute muscle strength was tested by hanging mice using their limbs. Mice were acclimated to the testing environment in their home cages for 1 hr prior, and body weights were measured prior to the start of testing. Three consecutive trials were then performed in 1 day. Mice were placed right-side-up on a standard cage lid with parallel metal bars, the lid was gently shaken three times to provoke grasping the bars, then the lid was slowly inverted to put mice in the hanging position. Trial duration maximum was 120 s. A 10 s inter-trial interval was given after falling. Mice remaining hanging up to the maximum time were inverted to right-side-up on the lid and given a 10 s interval before the next trial. Most WT mice can hang on for several minutes before losing grip and falling (Jones and Mormède, 2000), although each WT control cohort exhibited a wide range of latency to fall values. No sudden hypotonia was observed, and there was a spread in the range of Kcnma1 N999S/WT values. This observation suggests it unlikely that the hanging wire assay was a significant trigger for PNKD-like immobility in Kcnma1 N999S/WT mice, potentially due to the short duration compared to the full restraint stress used in Figure 5.

Statistics
Sample sizes were determined based on prior datasets, except the stress-induced behavioral assay where power calculation based on pilot data indicated sample sizes of 5 animals per group (effect size d=2.29 at 0.8 power with 0.05 α; G*power 3.1). Electrophysiology and behavioral data were tested for normality with Shapiro-Wilk normality test and either parametric or non-parametric statistics were analyzed in GraphPad Prism v9.02 (San Diego, CA). Outliers were determined by the ROUT method and were included in all datasets. Data are plotted as either mean ± SEM, or individual data points with median and inter-quartile range, as indicated in figure legends. The statistical test used for each dataset is indicated in the figure legend, and p<0.05 was considered significant. p-Values in figure legends are reported for post hoc tests when the main effect was p<0.05, or reported for the main effect, if p>0.05. Groups with three or fewer data points were not included in statistical analysis, as noted in legends. For parametric data, two-tailed, unpaired t-tests were performed with Welch's correction for unequal variance. For multiple comparisons, one-way ANOVAs were performed with Welch's correction followed by Dunnett's T3 post hoc test. Two-way repeated measures ANOVAs were performed with Geisser-Greenhouse correction followed by Bonferroni post hoc test for multiple comparisons (comparisons between genotypes across voltages). Mann-Whitney or Kruskal-Wallis followed by Dunn's multiple comparisons were used for non-parametric data.
In gene microarray studies, differential mRNA transcript expression was determined at a 2-fold change cutoff, with p>0.05 and false discovery rate, FDR = 0.99 using an ANOVA with an eBayes test was used (Ritchie et al., 2015) (Applied Biosystems Transcriptome Analysis Console (TAC) Software v4.0.1).

Additional files
Supplementary files • Transparent reporting form • Source code 1. Locomotor wheel activity script. Python code to calculate wheel speed and the number and duration of activity gaps.

Data availability
All data generated and analyzed during this study are included in the manuscript, or provided as source data files. Python code is provided as Source Code File 1.